Methods and compositions for thiol-acrylate based materials for 3d cell culturing in a microfluidic device

ABSTRACT

Provided are thiol-acrylate hydrogels and tunable cell culture materials including thiol-acrylate hydrogels, and methods of making thereof. Also provided are systems for forming three-dimensional cell culture scaffolds including the materials, and methods of culturing cells, including cancer cells, using thiol-acrylate hydrogels and tunable cell culture materials. The materials herein can be used in microfluidic droplet-generating devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 17/116,289 filed on Dec. 9, 2020, which application claims thebenefit of and priority to U.S. Provisional Application Ser. No.62/946,667, having the title “methods and compositions forThiol-Acrylate Based Materials for 3D cell culturing in a microfluidicdevice”, filed on Dec. 11, 2019, the disclosure of each which isincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under grant numberCBET 1511653, awarded by The National Science Foundation. The U.S.government has certain rights in the invention.

BACKGROUND

Two-dimensional (2D) cell culturing is the most common approach forcell-based research, despite evidence of variant gene expression,differences in cell-to-cell communication, a lack of accountability forthe spatial restriction of cells, and an inability to replicate masstransfer limitations including the existence of extracellular chemicalgradients. In the case of cancer, tumor cells grown in athree-dimensional (3D) environment have been shown to better replicatein vivo conditions including cell-to-cell interactions, metabolite anddrug transfer, and population heterogeneity — all characteristicslacking in modern 2D culturing approaches. Moreover, recent studies haveshown increased drug resistance in cancer cells cultured in a 3Denvironment compared to cells cultured in 2D when exposed to commonanti-cancer compounds. The tumor microenvironment is a complex systemconsisting of multiple types of cells (e.g., cancer, stromal, and immunecells) surrounded by extracellular matrix (ECM) deposition between thecells to facilitate cell-to-cell interaction, allow for the creating ofextracellular chemical gradients, and provide a scaffold to supportcellular growth. The ECM provides mechanical and chemical properties toproliferative cells; these properties can modulate cellular behavior.Such properties include stiffness, durability, temperature control,protein content, and the potential for degradation. Currently, the bestapproach for the 3D culture of cancer cells involves the growth andstudy of spheroids—three-dimensional, well-rounded cell aggregationsthat consist of multiple single cells. Several approaches exist togenerate these 3D spheroids including hanging droplet plates, spinnerflasks, and microfluidic devices including microwells and dropletgenerators. These different methods to generate spheroids take advantageof two approaches: (1) prevention of cellular settling by coating thesurface to facilitate the self-aggregation of cells into a spheroid and(2) utilizing polymer hydrogels to act a physical scaffold to allow for3D cellular proliferation. While the former approach can generate 3Dspheroids rapidly, they are highly heterogeneous and susceptible todisruption by fluid shear stress due to the lack of an ECM mimic toprovide structure and stability. Conversely, biological and synthetichydrogels can recapitulate the native ECM and encompass biophysicalproperties and biological functions found under in vivo conditions.

Hydrogels are widely used in biomedical research due to theirbiocompatibility, high water content, and high permeability fordifferent growth factors and metabolites. Biological hydrogels such ascollagen, Matrigel, and fibrin have specific biophysical and celladhesive properties to mimic the ECM; however, they suffer frombatch-to-batch variability and uncontrolled degradation which can affectreproducibility. Synthetic hydrogels like polyethylene glycol (PEG) andalginate offer excellent control and reproducibility but requireexternal initiation to fully crosslink the materials to produce theresulting hydrogel. In fact, PEG is currently one of the most widelyused monomers to synthesize hydrogels that is also FDA-approved.

Synthetic hydrogels are commonly synthesized using two approaches. Thefirst method involves the chemical crosslinking of the polymer usingphotopolymerization, click reactions, enzyme catalysis or Michael-typereactions. The second approach uses physically crosslinked hydrogelsthat form due to hydrophilic-hydrophobic or dipole-dipole interactions.While each of these approaches has found success for 3D cell cultureapplications, they are not without limitations. Physically-crosslinkedhydrogels are usually mechanically weaker and less stable thanchemically-crosslinked hydrogels. Photopolymerized hydrogels can resultin exogenous reactive radicals, reactive macromers, or initiators in thehydrogel which can adversely affect cell viability. In addition, rapidphotopolymerization processes can increase the local temperature withinthe hydrogel, which also decreases cell viability.

Despite advances in 3D cell culture research, there is still a scarcityof hydrogel systems that are biocompatible and biodegradable, arenon-cytotoxic, and have tunable properties such as stiffness, gelationtime, swelling ratio, and diffusion coefficient of small molecules.These needs and other needs are satisfied by the present disclosure.

SUMMARY

In accordance with the purpose(s) of the present disclosure, as embodiedand broadly described herein, the disclosure relates to thiol-acrylatehydrogels, methods of making the same, three-dimensional cell culturescaffolds comprising the same, systems for cell culture using the same,and methods of culturing cells, including cancer cells, using the same.

An aspect of the present disclosure includes a tunable cell culturematerial including a hydrogel, wherein the hydrogel is a product of areaction between a thiol and an acrylate. The thiol can includeethoxylated trimethylolpropane tri (3-mercapto-propionate) (ETTMP), andthe acrylate can include poly(ethylene glycol) diacrylate (PEGDA).

An aspect of the present disclosure includes a method for culturingcells. The method can include providing a tunable cell culture materialas above and seeding the cells in the hydrogel to form a seededhydrogel. The hydrogel can be contacted with a culture medium, then thecells can be allowed to grow for a period of from about 4 days to about17 days.

Another aspect of the present disclosure includes a system for 3D cellculture that includes a microfluidic droplet-generating device and athiol-acrylate hydrogel.

Other systems, methods, features, and advantages of the presentdisclosure will be or become apparent to one with skill in the art uponexamination of the following drawings and detailed description. It isintended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present disclosure, and be protected by the accompanying claims. Inaddition, all optional and preferred features and modifications of thedescribed embodiments are usable in all aspects of the disclosure taughtherein. Furthermore, the individual features of the dependent claims, aswell as all optional and preferred features and modifications of thedescribed embodiments are combinable and interchangeable with oneanother.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the present disclosure can be better understood withreference to the following drawings. The components in the drawings arenot necessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1A shows a synthesis scheme of thiol-acrylate hydrogels generatedby a base-catalyzed Michael addition at room temperature in accordancewith embodiments of the present disclosure and FIG. 1B shows a schematicrepresentation of ETTMP (three arm thiol) and PEGDA crosslinking (twoarm acrylate) resulting in bonding of the thiol to the acrylate when thereaction is stoichiometrically balanced (i) and when there is excessthiol (ii).

FIG. 2A shows FTIR characterization of PEGDA, ETTMP and thiol-acrylatehydrogel in accordance with embodiments of the present disclosure. FIG.2B shows a swelling profile of thiol-acrylate hydrogel in cell culturemedia at 37° C. in accordance with embodiments of the presentdisclosure. Hydrogel swelling ratio was monitored as a function ofweight percentage of polymer (8.5, 9.0, and 9.5%) and molar ratio ofthiol-to-acrylate groups (1.0 and 1.05) for 24 h.

FIG. 3 shows diffusion of bromothymol blue into the thiol-acrylatehydrogel according to the present disclosure. The mass transfer of thedye is modeled by an error function which was numerically fit to thedata using KaleidaGraph to calculate the diffusion coefficient (D). Datais for F2 at the 30 min time point which is representative of the masstransfer within all six hydrogels. (Inset) Images of the migrating frontof the bromothymol blue dye in F4 at 5 and 400 min.

FIGS. 4A-4B show the effect of pH on the degradation of covalentlycrosslinked thiol-acrylate hydrogels in accordance with embodiments ofthe present disclosure. All six formulations were incubated with (FIG.4A) DMEM (pH 8.05) or (FIG. 4B) PBS (pH 7.9) for up to 400 h at 37° C.

FIGS. 5A-5B show the calculation of rheological properties of sixthiol-acrylate hydrogels in accordance with embodiments of the presentdisclosure. The values for tan δ (FIG. 5A) and the complex shear modulusG* (FIG. 5B) were obtained from the bulk rheology measurements ofthiol-acrylate hydrogels taken for a frequency sweep of 0.682 to 62.8radians/second at 37° C.

FIG. 6 shows determination of 2D cell adhesion, growth, and viability onthiol-acrylate hydrogels in accordance with embodiments of the presentdisclosure. MDA-MB-231 cells were seeded onto F2 (A), F4 (B), and F1 (C)and allowed to grow for nine days at 37° C. in complete media.Representative images are shown for initial seeding on day 0 (i) alongwith brightfield (ii), FITC (iii, live cells), and rhodamine (iv, deadcells) on day 9. Scale bar is 100 μm. Images are representative ofduplicate. Images are representative of duplicate experiments.

FIG. 7 shows evaluation of 3D proliferation and viability of MDA-MB-231cells within thiol-acrylate hydrogels in accordance with embodiments ofthe present disclosure. M DA-M B-231 cells were seeded into F2 (A), F4(B), and F1 (C) and allowed to grow for 17 days at 37° C. in completemedia within the thiol-acrylate hydrogels. Representative images areshown for initial seeding on day 0 (i) along with brightfield (ii), FITC(iii, live cells), and rhodamine (iv, dead cells) on day 9. Scale bar is100 μm. Images are representative of triplicate experiments.

FIG. 8 shows evaluation of 3D spheroid formation of MCF7 cells withinthiol-acrylate hydrogels in accordance with embodiments of the presentdisclosure. MCF7 cells were seeded into F2 and allowed to grow for 12days at 37° C. in complete media within the thiol-acrylate hydrogels.Representative bright field images are shown for initial seeding on day0 and their growth over time on days 2, 3, 6, 8 and 12. Scale bar is 200μm in all images. Images are representative of duplicate experiments.

FIGS. 9A-9B show variation in gelation times for different formulationsof thiol-acrylate hydrogels in accordance with embodiments of thepresent disclosure. Three different weight percentages were examined,8.5% (F1/F2), 9.0% (F3/F4), and 9.5% (F5/F6), in addition to twodifferent molar ratios of thiol-to-acrylate group is 1.0 (FIG. 9A) and1.05 (FIG. 9B).

FIG. 10 shows the calculation of diffusion coefficient (D) in the sixhydrogel formulations in accordance with embodiments of the presentdisclosure. At each time point, the value of 4Dt was calculated from amathematical model of one-dimensional mass transfer fit to an errorfunction, and from the slope of each line D was calculated for eachformulation.

FIGS. 11A-11B show bulk rheology of thiol-acrylate hydrogels inaccordance with embodiments of the present disclosure. All measurementswere taken during a frequency sweep (0.682 to 62.8 radians/second) ofthe different formulations at 37° C. G′ is the storage modulus (orelasticity of hydrogel). (FIG. 11A) Three formulations (F1, F3, and F5)with a molar ratio of 1.0. (FIG. 11B) Three formulations (F2, F4, andF6) with a molar ratio of 1.05. Black lines and data points are for G′and red lines and data points are for G″.

FIG. 12 shows evaluation of 3D growth and viability of MDA-MB-231 cellswithin thiol-acrylate hydrogels in accordance with embodiments of thepresent disclosure. M DA-M B-231 cells were seeded into F3 (row A), F5(row B), and F6 (row C) and allowed to grow for 17 days at 37° C. incomplete media within the thiol-acrylate hydrogels. Representativeimages are shown for initial seeding on day 0 (i) along with brightfield(ii), FITC (iii, live cells), and rhodamine (iv, dead cells) on day 9.Scale bar is 100 μm. Images are representative of triplicateexperiments.

FIG. 13 shows quantification of cell viability in thiol-acrylatehydrogels in accordance with embodiments of the present disclosure. Thenumber of live and dead MDA-MB-231 cells were assessed during the 17-dayculture within the thiol-acrylate hydrogels using an automated Pythonalgorithm. A minimum of 175 cells were counted for each formulation ateach of the days that viability was assessed (days 4, 7, 11, and 17).

FIG. 14 shows evaluation of 3D spheroid formation of MCF7 cells withinthiol-acrylate hydrogels in accordance with embodiments of the presentdisclosure. MCF-7 cells were seeded into F4 (A) and F6 (B) and allowedto grow for 12 days at 37° C. in complete media within thethiol-acrylate hydrogels. Representative bright field images are shownfor initial seeding on day 0 and their growth over time in day 3, 8 and12. Scale bar is 200 μm. Images are representative of duplicateexperiments.

FIG. 15 shows a two-layer PDMS microfluidic droplet generator inaccordance with embodiments of the present disclosure. The bottom layercontains a trapping array, a microfluidic channel, and the top layer isa flat PDMS layer containing holes for inlets and an outlet.

FIGS. 16A-16D show generation of spheroid using a droplet generatorsystem (also referred to as a system for 3D cell culture) in accordancewith embodiments of the present disclosure. FIG. 16A is a top view ofthe droplet trapping array showing two inlets for carrier oil (1), cellsin the aqueous hydrogel (2), a flow-focusing junction (3), the droplettrapping array (4), and the single outlet (5). FIG. 16B is a side viewof the two-layer Polydimethylsiloxane (PDMS) device. FIG. 16C is aBrightfield image of MCF-7 cells trapped inside the hydrogel droplet inday and FIG. 16D shows spheroids on day 5. The scale bar is 150 μm.

FIGS. 17A-17C show generation of MCF-7 spheroid and changes of theirmorphology over time in absence of contentious media flow through adevice in accordance with embodiments of the present disclosure.Brightfield images of trapping array in day 0 (FIG. 17A), day 2 (FIG.17B), and day 6 (FIG. 17C) taken in 10× magnification under Leicamicroscope. The scale bar is 200 μm.

FIG. 18 shows a schematic representation of a gravity-driven media flowsetup for the microfluidic droplet generator device in accordance withembodiments of the present disclosure. mL plastic syringes were used asa reservoir (a) and collector (b). A closed 1.0 mL glass syringe (c) wasused to close the aqueous port of the device.

FIG. 19 shows a representation of gravity-driven media flow setup in anon-chip experiment in accordance with embodiments of the presentdisclosure.

FIGS. 20A-20E show on-chip viability of MCF-7 spheroids which wascultured on-chip for nine days before carried out live and dead stainingin accordance with embodiments of the present disclosure. Representativeimages are shown for brightfield images of trapping array in day 2 (FIG.20A) and day 9 (FIG. 20B). FITC (FIG. 20C, green indicates live cells),rhodamine (FIG. 20D, red indicates dead cells), and an overlay image(FIG. 20E) on day 9. The scale bar is 150 μm.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

Many modifications and other embodiments disclosed herein will come tomind to one skilled in the art to which the disclosed compositions andmethods pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the disclosures are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims. Theskilled artisan will recognize many variants and adaptations of theaspects described herein. These variants and adaptations are intended tobe included in the teachings of this disclosure and to be encompassed bythe claims herein.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure.

Any recited method can be carried out in the order of events recited orin any other order that is logically possible. That is, unless otherwiseexpressly stated, it is in no way intended that any method or aspect setforth herein be construed as requiring that its steps be performed in aspecific order. Accordingly, where a method claim does not specificallystate in the claims or descriptions that the steps are to be limited toa specific order, it is no way intended that an order be inferred, inany respect. This holds for any possible non-express basis forinterpretation, including matters of logic with respect to arrangementof steps or operational flow, plain meaning derived from grammaticalorganization or punctuation, or the number or type of aspects describedin the specification.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

While aspects of the present disclosure can be described and claimed ina particular statutory class, such as the system statutory class, thisis for convenience only and one of skill in the art will understand thateach aspect of the present disclosure can be described and claimed inany statutory class.

It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosed compositions andmethods belong. It will be further understood that terms, such as thosedefined in commonly used dictionaries, should be interpreted as having ameaning that is consistent with their meaning in the context of thespecification and relevant art and should not be interpreted in anidealized or overly formal sense unless expressly defined herein.

Prior to describing the various aspects of the present disclosure, thefollowing definitions are provided and should be used unless otherwiseindicated. Additional terms may be defined elsewhere in the presentdisclosure.

Definitions

As used herein, “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps, or components, or groups thereof.Moreover, each of the terms “by”, “comprising,” “comprises”, “comprisedof,” “including,” “includes,” “included,” “involving,” “involves,”“involved,” and “such as” are used in their open, non-limiting sense andmay be used interchangeably. Further, the term “comprising” is intendedto include examples and aspects encompassed by the terms “consistingessentially of” and “consisting of.” Similarly, the term “consistingessentially of” is intended to include examples encompassed by the term“consisting of.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a buffer,” “athiol,” or “an acrylate,” includes, but is not limited to, combinationsof two or more such buffers, thiols, or acrylates, and the like.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. Ranges can be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms a furtheraspect. For example, if the value “about 10” is disclosed, then “10” isalso disclosed.

When a range is expressed, a further aspect includes from the oneparticular value and/or to the other particular value. For example,where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to‘y’ as well as the range greater than ‘x’ and less than ‘y’. The rangecan also be expressed as an upper limit, e.g. ‘about x, y, z, or less’and should be interpreted to include the specific ranges of ‘about x’,‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, lessthan y′, and ‘less than z’. Likewise, the phrase ‘about x, y, z, orgreater’ should be interpreted to include the specific ranges of ‘aboutx’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’,greater than y′, and ‘greater than z’. In addition, the phrase “about‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’to about ‘y’”.

It is to be understood that such a range format is used for convenienceand brevity, and thus, should be interpreted in a flexible manner toinclude not only the numerical values explicitly recited as the limitsof the range, but also to include all the individual numerical values orsub-ranges encompassed within that range as if each numerical value andsub-range is explicitly recited. To illustrate, a numerical range of“about 0.1% to 5%” should be interpreted to include not only theexplicitly recited values of about 0.1% to about 5%, but also includeindividual values (e.g., about 1%, about 2%, about 3%, and about 4%) andthe sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%;about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and otherpossible sub-ranges) within the indicated range.

As used herein, the terms “about,” “approximate,” “at or about,” and“substantially” mean that the amount or value in question can be theexact value or a value that provides equivalent results or effects asrecited in the claims or taught herein. That is, it is understood thatamounts, sizes, formulations, parameters, and other quantities andcharacteristics are not and need not be exact, but may be approximateand/or larger or smaller, as desired, reflecting tolerances, conversionfactors, rounding off, measurement error and the like, and other factorsknown to those of skill in the art such that equivalent results oreffects are obtained. In some circumstances, the value that providesequivalent results or effects cannot be reasonably determined. In suchcases, it is generally understood, as used herein, that “about” and “ator about” mean the nominal value indicated ±10% variation unlessotherwise indicated or inferred. In general, an amount, size,formulation, parameter or other quantity or characteristic is “about,”“approximate,” or “at or about” whether or not expressly stated to besuch. It is understood that where “about,” “approximate,” or “at orabout” is used before a quantitative value, the parameter also includesthe specific quantitative value itself, unless specifically statedotherwise.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired modification of a physical property ofthe composition or material. For example, an “effective amount” of athiol refers to an amount that is sufficient to achieve the desiredimprovement in the property modulated by the formulation component, e.g.achieving the desired level of cell viability in the resulting hydrogel.The specific level in terms of wt % in a composition required as aneffective amount will depend upon a variety of factors including theamount and type of thiol, amount and type of acrylate, amount and pH ofbuffer, and end use of the hydrogel made using the composition.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

Unless otherwise specified, temperatures referred to herein are based onatmospheric pressure (i.e. one atmosphere).

As used herein, a “thiol” is an organic compound containing an —SHgroup. In one aspect, thiols are useful for synthesizing the hydrogelsdisclosed herein. In another aspect, a molecule useful herein can havetwo or more thiol groups. In one aspect, the thiol used herein isethoxylated trimethylolpropane tri (3-mercapto-propionate) (ETTMP):

As used herein, an “acrylate” is a salt, ester, or conjugate base ofacrylic acid and its derivatives. In another aspect, acrylates areuseful for synthesizing the hydrogels disclosed herein. In still anotheraspect, a molecule useful herein can have two or more acrylate groups.In one aspect, the acrylate used herein is poly(ethylene glycol)diacrylate (PEGDA):

The “Michael reaction” or “Michael addition” is nucleophilic addition toan α,β-unsaturated carbonyl compound. In one aspect, disclosed hereinare hydrogels formed by the Michael addition between a thiol and anacrylate. In another aspect, disclosed herein are hydrogels formed bythe Michael addition between ETTMP and PEGDA. In one aspect, the Michaeladdition disclosed herein requires a basic solution to proceed. In afurther aspect, the reaction product between one thiol group and oneacrylate group has the structure shown below:

As used herein, a “hydrogel” is a network of hydrophilic polymer chains.When the polymer chains are held together by crosslinks, a threedimensional solid can form. In one aspect, the hydrogel has structuralintegrity because of the presence of the crosslinks. In another aspect,a hydrogel is highly absorbent but does not dissolve in water. In oneaspect, disclosed herein are hydrogels formed from the Michael additionof thiols and acrylates.

As used herein, “crosslinking” refers to a process by which polymericchains are extended in a multidimensional manner. In one aspect,crosslinking results in a network or 3D structure. Crosslinking can becovalent or ionic, and restricts the ability of a polymer network tomove. In one aspect, crosslinking of a polymer that typically dissolvesor exists in a liquid state can form a solid or gel out of the polymer.In one aspect, disclosed herein are covalently crosslinked hydrogelsformed by the Michael addition of thiols and acrylates such as, forexample, ETTMP and PEGDA.

“Initiation” as used herein is the first step of the polymerizationprocess. In some aspects, a radical polymerization process initiateswhen an activating agent or external energy source (i.e., heat, UVirradiation) is used. In one aspect, the polymerization disclosed hereindoes not require a radical initiator or any source of external energy.In another aspect, initiation in the reactions disclosed herein occurswhen an aqueous basic solution is added to the reaction mixturesdisclosed herein.

“Photopolymerization” as used herein requires light (typicallyultraviolet but visible light can also be used in some instances) toinitiate and/or propagate a polymerization reaction. In some aspects,when living cells are incorporated into a hydrogel, the light used forphotopolymerization can damage, mutate, or kill the cells. In oneaspect, the polymerization process disclosed herein is not aphotopolymerization process and/or does not require an external lightsource.

As used herein, “gelation” is the formation of a gel from a polymericsystem. In a further aspect, branched or crosslinked polymers form asingle macromolecule; the point at which this occurs is the system's gelpoint. At the gel point, or when gelation has occurred, at least somefluidity is lost while viscosity increases. Meanwhile, “gelation time”as referred to herein is the time period required for a hydrogel toform. In one aspect, gelation time can be measured by inverting acontainer containing a composition as disclosed herein; when a bubblewill no longer traverse the contents of the container, gelation can besaid to have occurred. In a further aspect, gelation time can be tunedby varying the ratio of thiol to acrylate in the compositions disclosedherein.

“Rigidity” or “stiffness” as used herein is a property of a hydrogel. Inone aspect, when a hydrogel contains, serves as a scaffold for, orotherwise contacts cells, stiffness of the hydrogel can affect cellmotility and/or differentiation, as well as cell viability. In someaspects, G′ as discussed above is a measure of stiffness (or,conversely, elasticity) of the hydrogels disclosed herein. In someaspects, stiffness does not affect two-dimensional cell culture but ahigh degree of stiffness may be undesirable for three-dimensional cellculture on the hydrogels disclosed herein.

“Diffusion coefficient” is a measure of the amount of a substance thatpasses through each unit of cross section of a bulk material per unit oftime. Diffusion coefficient for a given substance will be differentdepending on the properties such as, for example, stiffness, of the bulkmaterial into which it is diffusing. In one aspect, diffusioncoefficient can be used as an approximation of mass transfer ofbiomolecules in the hydrogel formulations disclosed herein. In anotheraspect, mass transfer of biomolecules through the hydrogels disclosedherein is important to cell reproduction and viability, when thehydrogels are used for two-dimensional or three-dimensional cellculture.

“Swell ratio” or “swelling ratio” as used herein is inversely related tothe crosslinking density of a hydrogel. In some aspects, swell ratio isrelated to stiffness and diffusion rate of biomolecules into a hydrogel.In one aspect, swelling ratio can be calculated by dividing swollen gelrate at a specified time by dried gel weight.

“Shear storage modulus” (represented as G′) as used herein refers to theelastic (in phase) stress to strain ratio of a material in response toan oscillatory stress. Storage modulus relates to a material's abilityto store energy elastically and provides information about the amount ofstructure a material possesses and/or its resistance to deformation.

“Shear loss modulus” (represented as G″) as used herein refers to theviscous (out of phase) component of oscillatory stress. Loss modulusrelates to a material's ability to dissipate stress through heat andprovides information about the amount of energy dissipated in amaterial. When G′>G″, a material is primarily elastic. When G″>G′,externally applied forces cause a material to flow.

“Complex shear modulus” (represented as G*) relates to gel stiffnessunder dynamic conditions of deformation, regardless of whether thedeformation is elastic or viscous. In one aspect, G*=G′+iG″ where i isthe imaginary unit. In one aspect, G* is a material property thatrelates complex shear stress and complex shear strain.

“Delta (δ)” is the phase angle difference between applied stress anddeformation (strain). In one aspect, tan δ−G″/G′. In one aspect, tan δserves as an indication of the degree of energy dissipation or dampingof a material. A higher value (i.e., >1) for tan δ indicates that amaterial has more liquid-like properties, while a lower value (i.e., <1)for tan δ indicates that a material has more solid-like properties.

As used herein, “spheroid” refers to a three-dimensional, well-roundedaggregation consisting of multiple single cells. In one aspect, aspheroid can be used for three-dimensional culture for cancer cells.Numerous methods exist for generating spheroids including hangingdroplet plates, spinner flasks, and microfluidic devices. In one aspect,prevention of cellular settling can facilitate self-aggregation of cellsinto a spheroid. In an alternative aspect, a polymer hydrogel can beused as a physical scaffold to allow for three-dimensional cellularproliferation. In a further aspect, use of a polymer hydrogel scaffoldas in the techniques disclosed herein can mimic extracellular matrix andmore closely resembles conditions found in vivo.

“Extracellular matrix” is a three-dimensional network of macromolecules,typically proteins or glycoproteins (e.g., collagen, enzymes, fibrousproteins) and polysaccharides. Extracellular matrix (or ECM) isimplicated in cell communication, cell differentiation, and celladhesion. In one aspect, the hydrogel formulations disclosed herein canmimic the extracellular matrix.

“Biocompatible” materials, as used herein, are materials that are notharmful to living tissues and/or cells. In one aspect, biocompatiblematerials do not elicit an immune response. As used herein,“cytotoxicity” is a property of a compound wherein the compound willultimately cause cell death through any number of means including, butnot limited to, necrosis, lysis, apoptosis, or a cessation of growth anddivision. Cytotoxicity is dependent upon cell type; what is cytotoxic toone cell may not be to another. In one aspect, materials that arebiocompatible are not cytotoxic. In another aspect, the hydrogelsdisclosed herein are biocompatible and are not cytotoxic.

“Cell viability” refers to the quantification of the number of livingcells (still carrying out normal metabolic activities) in a cellculture. In one aspect, cell viability can be expressed as a percentageof the total number of cells in a sample, where some cells may be dead.In one aspect, cytotoxicity of a substance can be assessed by performinga cell viability assay.

Methods of Making Thiol-Acrylate Hydrogels

In one aspect, disclosed herein are hydrogels formed via a Michaeladdition of a thiol with an acrylate. In some aspects, startingmaterials can have multiple thiol and/or multiple acrylate functionalgroups in order to facilitate crosslinking and formation ofthree-dimensional structures. In one aspect, the thiol-bearing moleculecan have at least three thiol groups. In another aspect, theacrylate-bearing molecule can have at least two acrylate groups. In oneaspect, the thiol is ethoxylated trimethylolpropane tri(3-mercapto-propionate) (ETTMP) and the acrylate is poly(ethyleneglycol) diacrylate (PEGDA).

In one aspect, the PEDGA can have a number average molecular weight (Me) of from about 400 to about 4000 Da, or of about 400, 500, 600, 700,800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250,3500, 3750, or about 4000 Da, or a combination of any of the foregoingvalues, or a range encompassing any of the foregoing values. In oneaspect, the PEGDA has an M_(n) of about 700 Da.

In one aspect, different weight percentages of starting monomers can beused in order to fine-tune the properties (i.e., viscosity, swellingratio, gelation time, and the like) of the hydrogels disclosed herein.In one aspect, the weight percentage of monomers can be from about 7 toabout 11, or can be from about 8.5 to about 9.5, or can be about 7.7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25,10.5, 10.75, or about 11, or a combination of any of the foregoingvalues, or a range encompassing the foregoing values. In one aspect, theweight percentage of monomers is 8.5, 9, or 9.5.

In another aspect, different ratios of thiol to acrylate can be used inorder to fine-tune the properties of the hydrogels disclosed herein. Inanother aspect, the ratio of thiol groups to acrylate groups can be fromabout 0.95 to about 1.10, or can be from about 1 to about 1.05, or canbe about 0.95, 1.0, 1.05, or about 1.10, or a combination of any of theforegoing values, or a range encompassing the foregoing values. In oneaspect, the ratio of thiol groups to acrylate groups is 1.0 or is 1.05.

In still another aspect, disclosed herein is a process for preparingthiol-acrylate hydrogels. In a further aspect, the Michael additionreaction can proceed in any suitable buffer. In one aspect, the buffercan be an extracellular buffer or ECB. In a still further aspect, theECB is an aqueous solution of 5.036 mM HEPES, 136.89 mM NaCl, 2.68 mMKCl, 2.066 mM MgCl₂·6H 2 0, 1.8 mM CaCl₂·2H₂O, and 5.55 mM glucose.

In one aspect, the pH of the buffer used can be adjusted to a higher orlower value using an appropriate base or acid as needed. In one aspect,the buffer pH is adjusted to from about 7.5 to about 8.2, or to about7.5, 7.55, 7.6, 7.65, 7.7, 7.75, or about 7.8, or a combination of anyof the foregoing values, or a range encompassing the foregoing values.In one aspect, the buffer pH is adjusted to 7.66. In any of theseaspects, a slightly basic pH for the buffer may be required forinitiation of the Michael reaction. In a further aspect, the buffer pHis adjusted with NaOH, KOH, or another commonly-used base. In oneaspect, the buffer pH is adjusted with 5 mM NaOH. Advantageously,because the hydrogels described herein are crosslinked by abase-catalyzed Michael addition, the hydrogel does not require anyexternal initiation.

In one aspect, following preparation and pH adjustment of the buffer,PEGDA can be added to the buffer and mixed by any suitable means. In oneaspect, the PEGDA solution in buffer is vortexed for from about 10seconds to about 30 seconds, or for about 10, 15, 20, 25, or about 30seconds, or a combination of any of the foregoing values, or a rangeencompassing any of the foregoing values. In one aspect, the PEGDAsolution in buffer is vortexed for about 15 seconds.

In another aspect, following complete dissolution of PEGDA in thebuffer, ETTMP is added to the PEGDA-buffer mixture. In another aspect,the solution of PEGDA, buffer, and ETTMP can be mixed by any suitablemeans. In one aspect, the solution of PEGDA and ETTMP in buffer can bevortexed for from about 15 seconds to about 90 seconds, or can bevortexed for about 15, 30, 60, 75, or about 90 seconds, or a combinationof any of the foregoing values, or a range encompassing any of theforegoing values. In one aspect, the solution of PEGDA and ETTMP inbuffer is vortexed for about 1 minute.

In any of the above aspects, following mixing of the PEGDA and ETTMP inbuffer, the solution can be left undisturbed for a sufficient period toachieve gelation.

Properties of the Thiol-Acrylate Hydrogels Gelation Time

In one aspect, gelation time for the hydrogels disclosed herein can befrom about 10 to about 300 minutes, or from about 25 to about 180minutes, or can be about 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,290, 295, or about 300 min, or a combination of any of the foregoingvalues, or a range encompassing any of the foregoing values. In oneaspect, the gelation time is 10 minutes, 25 minutes, 40 minutes, 180minutes, or 300 minutes. In another aspect, gelation time correlateswith monomer percentage and ratio of thiol to acrylate. In one aspect, afaster gelation time may result in the formation of a stiffer hydrogel.Thus, in one aspect, a somewhat slower gelation time, resulting in ahydrogel with less stiffness, is preferred for three-dimensional cellculture applications. In some aspects, a molar ratio of components of1.05 (i.e., ratio of thiol groups to acrylate groups) as disclosedherein can lead to a gelation time of from about 25 to about 40 minutes,or in some cases from about 25 to about 60 minutes. In other aspects, amolar ratio of components of 1.0 can lead to a gelation time of fromabout 30 to about 150 minutes, or from about 25 to about 180 minutes insome cases.

Swell Ratio

In another aspect, swelling ratio for the hydrogels disclosed herein canbe determined by weighing the hydrogel when dried, incubating thehydrogel in culture medium (such as, for example, ECB), and weighing therehydrated hydrogel, then taking the ratio of rehydrated hydrogel todried hydrogel. In one aspect, the hydrogels disclosed herein can bedried by leaving them out overnight at room temperature. In anotheraspect, incubation in culture medium typically takes place at about 37°C. In one aspect, swell ratio after 4 hours of rehydration can be fromabout 2 to about 5, or can be from about 2.5 to about 4.5, or can beabout 2, 2.5, 3, 3.5, 4, 4.5, or about 5, or a combination of any of theforegoing values, or a range encompassing any of the foregoing values.In another aspect, swell ratio after 24 hours of rehydration can be fromabout to about 8, or can be from about 1 to about 7, or can be about0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, or about 8, ora combination of any of the foregoing values, or a range encompassingany of the foregoing values.

Diffusion Coefficient

In another aspect, diffusion coefficient for biomolecules can becalculated for the hydrogels disclosed herein. In a further aspect,diffusion coefficient for a model system can be used to approximatediffusion and/or mass transfer behavior for other molecules importantfor applications such as, for example, cell culture.

In one aspect, an example molecule can be dissolved in water or in ECBor another suitable buffer. In one aspect, the molecule has a color insolution that can be observed by eye and/or photographed with a camera.In a further aspect, the molecule can be at a concentration of about 1%(w/w) in the water. In a still further aspect, the solution of moleculein water can be dropped on a hydrogel formed in a plastic cuvette. Insome aspects, diffusion of the molecule can be photographed at anydesired interval. Photographic data can be quantified such that it isassumed gray level intensity is proportional to model moleculeconcentration; in one aspect, gray level intensity versus verticalposition can be modeled to determine diffusion coefficient. Exemplaryprocedures for calculating diffusion coefficient can also be found inthe Examples.

In one aspect, diffusion coefficient can be from about 5 to about10×10⁻⁸ cm²/s, or can be about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,or about 10×10⁻⁸ cm²/s, or a combination of any of the foregoing values,or a range encompassing any of the foregoing values. In another aspect,diffusion coefficient can be about 6.3, 6.5, 6.7, 7.2, 9.4, or 9.5×10⁻⁸cm²/s.

Degradation of Hydrogels

In some aspects, it may be valuable to know the conditions under whichthe hydrogels disclosed herein degrade, and the extent to which theydegrade, as well as over what time period this degradation can happen.In one aspect, hydrogels can be weighed prior to degradation experimentsto establish an initial weight. The hydrogels can then be incubated at37° C. in 5% CO 2 in a suitable buffer in a humidified incubator for adesired period of time, the surrounding solution removed, and thehydrogels weighed again to obtain a degraded weight. If additionaldegradation studies are desired, in one aspect, the solution can beadded back to the weighed samples and they can be placed in theincubator for an additional time period.

In any of the above aspects, a high pH buffer or medium can be used fordegradation studies. In one aspect, ester hydrolysis rates increase athigher pH levels. In one aspect, the buffer or medium can be Dulbecco'sModified Eagle Media (DMEM) with a pH of 8.05. In an alternative aspect,the buffer or medium can be phosphate-buffered saline (PBS) with a pH of7.9. In some aspects, the buffer or medium can contain an antibioticsuch as, for example, penicillin or streptomycin to prevent bacterialcontamination during the degradation study. In one aspect, an amount ofDM EM or PBS as described above can be placed on the hydrogel and thehydrogel can then be placed in the incubator under the conditionspreviously described.

In one aspect, when the buffer or medium is DMEM, complete degradationof the hydrogels takes from about 40 to about 200 hours, or from about60 to about 150 hours, or about 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, or about 200 hours, or a combinationof any of the foregoing values, or a range encompassing any of theforegoing values. In another aspect, when the buffer or medium is PBS,complete degradation of the hydrogels takes from about 50 to about 500hours, or from about 100 to about 425 hours, or about 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, orabout 500 hours, or a combination of any of the foregoing values, or arange encompassing any of the foregoing values. As used herein, completedegradation means that the relative weight of the material decreases tono greater than one millionth of an original weight of the material orabout zero. Additional exemplary procedures for assessing hydrogeldegradation rates can be found in the Examples.

Rheology

In one aspect, the rheological properties of the hydrogels disclosedherein can be measured from bulk sample deformation using a rheometer.In a further aspect, rheological properties can be determined by anysuitable method able to subject the hydrogels to oscillatory stress anddetermine their response. In one aspect, small amplitude oscillatoryshear can be implemented. In a further aspect, a parallel disc rheometercan be used. In a further aspect, the parallel discs can be 8 mm indiameter. In any of these aspects, the rheological properties can bedetermined at any desired temperature. In one aspect, since it isdesired to use the hydrogels as cell culture media for human cancercells, rheological properties can be determined at 37° C. so as to moreaccurately reflect conditions in the human body. In a further aspect, afrequency range of from about 0.65 to about 65 rad/s can be used, orfrom about 0.682 to about 62.8 rad/s can be used, or about 0.65, 0.75,0.85, 0.95, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, orabout 65 rad/s can be used, or a combination of any of the foregoingvalues, or a range encompassing any of the foregoing values. In anotheraspect, a constant shear strain amplitude of from about 7% to about 13%,or of about 7, 8, 9, 10, 11, 12, or about 13% can be used, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, rheological properties can bemeasured under a constant shear strain amplitude of about 10%.

In a further aspect, G* and tan δ as described previously can bemeasured as proxies for material stiffness. In some aspects, when0≤tan≤δ, a material is purely elastic. In other aspects, when tan δ>1, amaterial acts as a viscous liquid. In any of these aspects, when angularfrequency is between about 1 and 100 rad/s, tan δ is from about 0.01 toabout 10, or is about 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, or about 10, ora combination of any of the foregoingvalues, or a range encompassing any of the foregoing values. In anotheraspect, when angular frequency is between about 1 and 100 rad/s, G* isfrom about 10 to about 1000 Pa, or is about 10, 25, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, or about 1000 Pa, or a combination of any of the foregoing values,or a range encompassing any of the foregoing values. Additionalexemplary procedures for measuring and/or calculating rheologicalproperties can be found in the Examples.

Cell Culture Using the Thiol-Acrylate Hydrogels Cell Viability Assays

In one aspect, in either two-dimensional or three-dimensional cellculture methods using the hydrogels disclosed herein, cell viability canbe assessed using any method known in the art. In one aspect, a cellviability assay includes using Calcein AM to identify living cells. In afurther aspect, Calcein AM is a non-fluorescent, hydrophobic compoundthat permeates intact, living cells. Further in this aspect,intracellular esterases produce calcein, a hydrophilic fluorescentcompound that is retained in the cytoplasm. In another aspect, ethidiumhomodimer-1 can be used to identify dead cells. In one aspect, a 3.5 μMsolution of Calcein AM in ECB is used to assess cell viability. Ethidiumhomodimer-1 (EthD-1) is a stain with a high affinity for nucleic acidsthat emits red fluorescence once bound to a nucleic acid. In one aspect,EthD-1 cannot enter a living cell because the living cell does not havea compromised membrane. In another aspect, dead cells have disruptedmembranes, thus giving EthD-1 the ability to enter and bind to DNA. Inone aspect, EthD-1 can stain dead cells regardless of the method of celldeath (e.g., lysis, apoptosis, etc.). In one aspect, a 3.85 μM solutionof EthD-1 in ECB is used to assess the number of dead cells present in asample.

In another aspect, cells can be cultured for any length of time desiredprior to assessing cell viability. In one aspect, cells are cultured at37° C. with 5% CO₂ in a humidified incubator for up to 9 days. Furtherin this aspect, complete media can be replenished at intervals to ensuresufficient nutrients were present for the cells. In any of the aboveaspects, to assess viability, staining solution can be added to thesamples where the assessment is desired, the mixture of sample andstaining solution can be incubated for up to 70 minutes in the dark, andvisualization can be accomplished using fluorescence microscopy. In someaspects, a microscope camera can be used with a FITC filter (green, forlive cells) or a rhodamine filter (red, for dead cells).

In any of the above aspects, all vortexing of hydrogel preparations isconducted prior to the addition of cells to avoid problems with cellularshearing. If desired, in one aspect, a cell/hydrogel suspension can begently mixed using a micropipette to ensure a homogeneous distributionof cells.

Two-Dimensional Cell Culture

In one aspect, two-dimensional cell culture can be used as an initialmeasure of hydrogel biocompatibility and/or cytotoxicity to avoid issuesregarding mass transfer of nutrients. In one aspect, in two-dimensionalcell culture situations, a 1.05 molar ratio of thiol groups to acrylategroups results in a sample wherein from about 90 to about 100% of cellsremain viable after 4, 7, 11, 17, or 24 days. In another aspect, fromabout 95 to about 97% of cells remain viable, or about 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or about 100% of cells remain viable, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In another aspect, after about 36-48 hours,triple negative breast cancer cells (MDA-MB-231) plated on a 1.05 molarratio hydrogel migrated away from the seeding site and adopted themorphology typically seen with this cell type in other culturescenarios.

In another aspect, in two-dimensional cell culture situations, a 1.0molar ratio of thiol groups to acrylate groups results in a sample whereonly from about 1 to about 10% of cells remain viable after 4, 7, 11, or17 days. In another aspect, about 1 to about 5% of cells remain viable,or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10% of cells remain viable,or a combination of any of the foregoing values, or a range encompassingany of the foregoing values. In one aspect, a 1.0 molar ratio of thiolto acrylate groups can prevent cell attachment to the hydrogel. Inanother aspect, a 1.0 molar ratio of thiol to acrylate groups may beconsidered cytotoxic. In another aspect, triple negative breast cancercells (MDA-MB-231) plated on a 1.0 molar ratio hydrogel remained at thesite of deposition and never adopted the morphology associated with thiscell type.

In one aspect, weight percentage of hydrogel monomers does not impactcell viability, morphology, or spreading in two-dimensional culture.

Three-Dimensional Cell Culture

In one aspect, similarly to the two-dimensional results, none of thehydrogels with a 1.0 molar ratio of thiol to acrylate is able to sustaincell viability for 17 days in three-dimensional cell culture conditions.Without wishing to be bound by theory, this may be due to fasterdegradation times of these hydrogel formulations; rapid degradation maybe incompatible with cell spreading on hydrogels. In a further aspect,unlike with the two-dimensional results, weight percentage of hydrogelmonomers can have an impact on cell viability in three-dimensional cellculture conditions. In one aspect, cells seeded within formulationshaving lower weight percentages (e.g., 8.5% and 9%) grew and remainedviable after 17 days of culture. In some aspects, in these formulationscells formed aggregates with a mixture of living and dead cells. In oneaspect, about 60% of cells are viable after 17 days.

In another aspect, cells plated on hydrogels with higher weightpercentages (e.g., 9.5%) may exhibit lower viability than cells platedon lower weight percentage hydrogels. In one aspect, the 9.5% hydrogelsare stiffer than those with lower weight percentages. Further in thisaspect, stiffer hydrogels may not facilitate growth conditions necessaryfor cell viability.

In any of the above aspects, isolated cancer cells were less likely tobe proliferative and more likely to be dead by the end of the cultureperiod. In another aspect, cells seeded near to one another in groupswere more likely to survive under any of the experimental conditionsdescribed herein. In one aspect, cells can be seeded on top of thehydrogels. In an alternative aspect, cells can be seeded inside thehydrogels. Further in this aspect, when cells are seeded inside thehydrogels, the cells can survive for from at least 4 to at least 12days, or for 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, or a combination ofany of the foregoing values, or a range encompassing any of theforegoing values.

In one aspect, three-dimensional cell culture as disclosed herein canoccupy any standard well plate or culture dish intended for cellculture. In one aspect, a 6-well, 12-well, 24-well, or 48-well plate canbe used, or a culture dish of any diameter including 35 mm, 60 mm, or100 mm. Further in this aspect, gel volume can be from about 100 μL toabout 10 mL, or can be about 100, 200, 300, 400, 500, 600, 700, 800, or900 μL, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 mL, or acombination of any of the foregoing values, or a range encompassing anyof the foregoing values. In one aspect, gel volume can be about 100 μL,about 200 μL, about 3 mL, or about 6.5 mL.

Microfluidic Device for Cell Culture

In some aspects, the microfluidic device includes a microfluidic droplettrapping array. In one aspect, the microfluidic droplet trapping arraycan generate 100 — 300 μm aqueous droplets containing single cellsencapsulated by the thiol-acrylate hydrogels disclosed herein.Advantageously, polymerization of the hydrogel takes place duringdroplet generation and trapping, resulting in a rapid encapsulation ofthe cells. In a further aspect, the droplets disclosed herein can beused for drug screening and/or basic research into processes occurringin diseases such as, for example, cancer and heart disease.

In some aspects, the microfluidic droplet-generating device can be usedas system for 3D cell culture that includes a thiol-acrylate hydrogelfor cell seeding.

In one aspect, the microfluidic droplet-generating device includes adroplet trapping array located below a flow channel. The trapping arraycan include a plurality of circular traps having a diameter of about 70μm to about 300 μm. For example, the diameter of each circular trap canbe about 70 μm, about 150 μm, or about 300 μm. In particular aspects,the trapping array can have about 785 traps having a diameter of 70 μmeach, about 990 traps having a diameter of 150 μm each, or about 450traps having a diameter of 300 μm each. Other sizes and quantities canbe envisioned by one of ordinary skill in the art.

In some aspects, the microfluidic droplet-generating device includes aflat layer having two inlets and an outlet. The flat layer is above abottom PDMS device layer that includes microfluidic flow channels and atrapping array, such the that the trapping array is provided below theflow channels.

In one aspect, the microfluidic device has two inlets, one for oil andanother for unpolymerized hydrogel containing cells. Further in thisaspect, flow from each inlet can be adjusted to select a droplet size.In a further aspect, following droplet generation, growth media can beused to flush oil out of the device and to transfer droplets to atrapping array where further experiments can be conducted while thedroplets are maintained in place.

Cell Culture in a Microfluidic Device using the Thiol-Acrylate Hydrogels

Although spheroids are useful for three-dimensional cell culture, theycan still exhibit heterogeneity in terms of size and cellulardistribution when generated from multiple cells. In one aspect,disclosed herein is a method for generating large numbers ofthree-dimensional spheroids from a single progenitor cell. In a furtheraspect, the method makes use of microfluidic devices to generate uniformspheroids. Advantageously, the spheroids generated by the methodsdescribed herein can exhibit greater homogeneity than spheroidsgenerated by existing methods. In a still further aspect, athiol-acrylate based hydrogel as described above in conjunction with amicrofluidic device can serve as the basis for rapid, facile generationof three-dimensional spheroids.

In one aspect, cells can occupy from about 10% to about 20% of thehydrogel volume, or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or about 20% of the hydrogel volume, or a combination of any of theforegoing values, or a range encompassing any of the foregoing values.

In some aspects, the hydrogels used in microfluidic devices canincorporate biomolecules such as, for example, RGD peptide (i.e.,arginylglycylaspartic acid), which is in some aspects responsible forcell adhesion to the extracellular matrix, or hyaluronic acid, which canmodulate the flowability and/or viscosity of the extracellular matrix.In a further aspect, incorporation of these biomolecules betterrecreates the three-dimensional cellular environment for cell culturepurposes.

In some aspects, a method for generating spheroids includes using thehydrogels and microfluidic droplet-generating devices described herein.Air can be removed from the device by injecting a non-flammable heattransfer fluid (referred to herein as an oil) into the inlet and flowingthrough the device. In some aspects, the oil can be Novec™ 7500 (3M™). Amaterial including thiol acrylate hydrogel mixed with cells of interestis injected into an inlet of the device until the traps in the trappingarray are filled. Excess material is flushed from the device by a secondflowing of oil, and the material is allowed to polymerize. In someaspects, the thiol acrylate hydrogel is a 8.5 wt. % thiol acrylatehydrogel. A culture growth media is flowed into the device to flushremaining oil and to sustain cell growth and proliferation. The growthmedia can be flowed for about 24 to 48 hours. In some aspects, agravity-flow setup can be used to allow growth media to continually flowover the trapped droplets (a.k.a. spheroids). In some aspects, thegrowth media can be paused for a period of about 24-48 hours, and thenresumed at intervals for periodic feeding (e.g. in about 24-hourintervals).

The method is tunable based on the desired outcome. Larger spheroids(e.g. about 50-100 or more cells) can be generated faster using largertrapping arrays, but may be more heterogeneous due to the larger span ofgenotypes arising from a larger number of initial cells. Smallerspheroids (e.g. about 4 to about 10 cells in a 150 um droplet) that aremore homogeneous can be generated, but the process takes more time forthe smaller spheroid to grow into a larger spheroid. The generatedspheroids can be used in an array of on-chip or other experiments.

Advantageously, the process for generating spheroids is rapid. Dropletsin the aqueous phase can be achieved within 10 minutes from the mixtureof the unpolymerized hydrogel and cells to droplet formation in thedevice. The completion of gelation (polymerization) of the aqueousthiol-acrylate reaction mixture occurs in the trapping array in about 35minutes.

Now having described the aspects of the present disclosure, in general,the following Examples describe some additional aspects of the presentdisclosure. While aspects of the present disclosure are described inconnection with the following examples and the corresponding text andfigures, there is no intent to limit aspects of the present disclosureto this description. On the contrary, the intent is to cover allalternatives, modifications, and equivalents included within the spiritand scope of the present disclosure.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of thedisclosure and are not intended to limit the scope of what the inventorsregard as their disclosure. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Materials and Methods used in Examples Chemicals

Polyethylene glycol diacrylate (PEGDA) (M_(n) 700) was purchased fromSigma Aldrich. Ethoxylated trimethylolpropane tri(3-mercaptopropionate)1300 (ETTMP 1300) was generously donated by Evans Chemetics LP.Extracellular buffer (ECB: 5.036 mM HEPES, 136.89 mM NaCl, 2.68 mM KCl,2.066 mM MgCl₂·6H₂O, 1.8 mM CaCl₂·2HO, and 5.55 mM glucose) was used asthe solvent for hydrogel synthesis. 5 M NaOH was added to the buffer tobring it to a final pH of 7.66. Unless otherwise stated, all otherreagents were obtained from Sigma Aldrich.

Cell Culture and Reagents

MDA-MB-231 is a triple-negative breast cancer cell line that has beenshown to be very aggressive and drug resistant, whereas MCF7 is anER-positive breast cancer cell line that is less aggressive and moresusceptible to therapy. Both cell lines were maintained with Dulbecco'sModified Eagle Medium (DMEM) (Corning) supplemented with 10% v/v HyCloneCosmic Calf Serum (VWR Life Sciences Seradigm), 1% MEM Essential AminoAcids (Quality Biological Inc.), 1% MEM Non-Essential Amino Acids(Quality Biological Inc.), 1 mM sodium pyruvate (Thermo FisherScientific), and 6 μL insulin/500 mL media (Insulin, Human Recombinantdry powder, Sigma Aldrich). Cells were maintained in T-75 flasks in ahumidified incubator at 37° C. and 5% v/v CO₂. Cells were subculturedwhen confluent by first washing the cells with 1× phosphate bufferedsaline (PBS: 137 mM NaCl, 10 mM Na₂HPO₄, 27 mM KCl, and 1.75 mM KH₂PO₄at pH 7.4) and then detaching the cells with Trypsin-EDTA (Corning) andMCF-7 was with 1× PBS (137 mM NaCl, 10 mM Na₂HPO₄, 27 mM KCl, and 1.75mM KH₂PO₄ at pH 7.4) prior to re-seeding into a new T-75 flask.

Example 2 Exemplary Hydrogel Synthesis

Three different weight percentages (8.5, 9, and 9.5%) of thiol-acrylatehydrogels, each with two different molar ratios of thiol groups toacrylate groups (1.0 and 1.05) were synthesized at room temperature(herein referred to as formulations 1-6, Table 1). To initiate thereaction, ˜13-20 μL of 5 M NaOH (depending upon the total volume of thehydrogel) was added to ECB which made the reaction basic enough forMichael addition (FIG. 1A); The PEGDA was then added to the ECB andvortexed for 15 seconds to disperse it in the buffer solution. Finally,the ETTMP was added and the complete reaction solution was vortexedvigorously for one minute and left undisturbed to complete the reaction.Gelation time was measured by regularly inverting the hydrogel in atube. The gelation time was identified as the time at which a bubblewould no longer rise in the hydrogel.

TABLE 1 Composition and Gelation Time of Different Thiol-AcrylateHydrogel Formulations Molar Ratio of Thiol Gelation Time FormulationWeight % of Gel to Acrylate Group (min) F1 8.5 1.0 150 F2 8.5 1.05 40 F39 1.0 80 F4 9 1.05 30 F5 9.5 1.0 30 F6 9.5 1.05 25

NaOH was used to deprotonate a thiol group (—SH) into a thiolate (—S—),which then allows for the addition of an acrylate group. Increasing theamount of the thiol in the reaction mixture did maximize the reactionprobability between thiol and acrylate. Additionally, adding excessthiol resulted in some uncrosslinked thiol groups (FIG. 1B, red armsshown in (ii), which may increase the hydrogel mesh size.

One requirement for an optimal hydrogel for 3D cell culture applicationsis the ability to quickly polymerize to allow for the addition ofculture media to ensure no reduction in cellular viability. It was foundthat the gelation time of the thiol-acrylate hydrogels variedsignificantly by tuning the thiol-to-acrylate molar ratios and relativeweight percentages of the two monomers (Table 1 and FIGS. 9A-9B). Thegelation times were found to be inversely related to the weightpercentages of the monomers present in the hydrogel. Increasing theweight percent from 8.5 to 9.5% resulted in a 5-fold decrease ingelation time from 150 min (F1) to 30 min (F5). This effect was not asprominent in the presence of excess thiol with only a 1.6-fold decreasein gelation time from 40 min (F2) to 25 min (F6).

The molar ratio of thiol-to-acrylate groups was found to be a factor inseveral aspects of the hydrogel. A large reduction in gelation time wasobserved by changing the ratio from 1.0 to 1.05; however, this effectwas diminished with increasing monomer content. Additionally, alteringthe molar ratio from 1.0 to 1.05 resulted a higher degree of cellularviability and growth during 3D cell culture. Changing the molar ratiofrom 1.0 to 1.05 resulted in the two components actually reaching astoichiometric ratio. The data presented here indicated that aninexpensive thiol (ETTMP) and acrylate (PEGDA) are able to generatestabile, noncytotoxic hydrogels without any need for purification of thechemicals.

Example 3 Characterization of Exemplary Hydrogels

FTIR characterization

Thiol-acrylate hydrogel samples were dehydrated with acetone by placinga small amount of sample in a glass vial where it was serially soaked in25, 50, 75, and 100% acetone for 30 min in each. Later, the samples weredried at room temperature overnight in negative 1 atm pressure. Finally,the FTIR spectra of monomers (PEGDA, ETTMP) and dried hydrogel werecollected using a Bruker Tensor 27 FTIR spectrophotometer equipped witha Pike Miracle single bounce diamond attenuated total reflectance (ATR)cell. FTIR data confirmed that thiol-acrylate h drogels were formed byreacting thiols with acrylate groups. However, in thiol-acrylatehydrogel spectra (FIG. 2A), some important IR bands are absent that werepresent in the spectra of monomers. Such bands are 2,560, 1,635, 1,408,990, and 810 cm⁻¹, which are responsible for S—H stretching (thiol), C═Cstretching (acrylate), ═CH2 bending, ═CH2 wagging, and ═CH2 twisting,respectively, are absent in thiolacrylate hydrogel spectra.

Swell Ratio Determination

The swelling ratio is an important parameter of a hydrogel forbiological applications as this ratio is inversely related to thecrosslinking density of a hydrogel, which ultimately affects stiffnessand diffusion rate of biomolecules into the hydrogel. Hydrogel weightswere taken under three conditions: (i) immediately after the synthesisof the hydrogel, (ii) after the hydrogel was dried, and (iii) after thehydrogel was rehydrated with buffer to induced swelling. After completegelation, the total weight of the hydrogel was obtained. Then thehydrogel was dried overnight at room temperature and weighed again(W_(d)). Then the hydrogel was rehydrated by immersing it in culturemedia followed by incubation at 37° C. for 24 hours. The sample weightof the rehydrated hydrogel was collected after 4 and 24 hours ofincubation. The equilibrium weight swelling ratio (Q) was calculatedusing Eqn. 1:

$\begin{matrix}{Q = \frac{W_{s}}{W_{d}}} & {{Eqn}.1}\end{matrix}$

where W_(s) is the swollen gel weight at a specified time interval andW_(d) is the dried gel weight.

F1, F3, and F5 (1.0 molar ratio of thiol-to-acrylate) exhibited maximumswelling (equilibrium swelling) in culture media between 4 to 5 hours,whereas F2, F4, and F6 (1.05 molar ratio) reached their maximum swellingwithin in 24 hours (FIG. 2B). Among all six formulations, F2 exhibitedthe highest swelling ratio (close to 6).

Diffusion Coefficient Approximation

In addition to the transport of water into the hydrogel, the diffusivemass transfer of biomolecules is essential for proper cell proliferationand viability during 3D cell culture applications. To approximate themass transfer of biomolecules into the thiol-acrylate hydrogels, thediffusion coefficient of bromothymol blue was measured (FIG. 3 ). Asolution of 1 wt % bromothymol blue was made in deionized (DI) water.For this experiment, each hydrogel formulation was synthesized in aplastic cuvette (10×10×48 mm). Once the hydrogel solidified, 200 μL of1% dye solution was carefully placed on top of the hydrogel. Images werecollected every 60 min for 400 min using a Nikon D3200 (072 Dill TAMRON18-270 mm). The grey level intensity versus vertical position in thecuvette was analyzed using ImageJ (NIH). The analysis assumed that thegrey level intensity was proportional to dye concentration. The greylevel profile along the vertical axis was fit to Eqn. 2, where y isposition, t is time, and D is the diffusion coefficient:

$\begin{matrix}{{C\left( {y,t} \right)} = {{\frac{1}{2}{erf}\left( \frac{x}{2\sqrt{Dt}} \right)} + \frac{1}{2}}} & {{Eqn}.2}\end{matrix}$

At each time point, the grey level intensity profile was fitted for2√{square root over (Dt)} using KaleidaGraph 4.5 (Reading, PA). Thevalues of 4Dt were then plotted against time to generate a line wherethe 0.25×slope provided the value of the diffusion coefficient.

The migrating front of the bromothymol dye was observed for 400 min atvarying time points (FIG. 3 , inset) and then fit to an error function,which approximates one-dimensional mass transfer, at each time point.Values relating the diffusion coefficient (D) to time were plottedresulting in a linear relationship (FIG. 10 ), which allowed for thedetermination of the six diffusion coefficients (Table 2). The valuesfor D were found to vary between 6.3×10⁻⁸ cm²/s to 9.5×10⁻⁸ cm²/s forthe six different hydrogel formulations (Table 2). These findingsconfirm the mass transfer of water and biomolecules into thethiol-acrylate hydrogels. In the experiments performed here using cancercell culture, complete medium was added after hydrogel synthesis, whichresulted in hydrogel swelling. Similarly, during experimentation, thehydrogels were incubated at 37° C. Both of these conditions can increasethe actual value of the diffusion coefficient over the measured value,which can explain how the cells were able to grow and thrive for up to17 days in the hydrogels. This is evidenced by the presence of viableMDAMB-231 cells of after 17 days in F2 and F4, and also the spheroidformation of MCF 7 cells and an increase in spheroid diameter from Day 0to Day 12 in F2, F4, and F6.

TABLE 2 Calculated Diffusion Coefficients of Different Thiol-AcrylateHydrogel Formulations Using Bromothymol Blue (MW 624 g/mol) FormulationDiffusion Coefficient (cm²/s) F1 9.4 × 10⁻⁸ F2 6.5 × 10⁻⁸ F3 9.5 × 10⁻⁸F4 6.7 × 10⁻⁸ F5 7.2 × 10⁻⁸ F6 6.3 × 10⁻⁸

Determination of Thiol-Acrylate Hydrogel Degradation

It has been reported that hydrogel degradation can occur due to esterhydrolysis. To investigate the pH dependence of hydrogel degradation,all six formulations were incubated with either DMEM (the basal mediafor cell culture, pH 8.05) or PBS (a phosphate buffered solution, pH7.9) at 37° C. (FIG. 6 ). Hydrogel degradation was monitored using agravimetric method. For the degradation study, each hydrogel formulationwas synthesized in 8×40 mm glass vials. Once all the hydrogels weresolidified, the initial weight (Wo) was measured. Then either 500 μL ofDMEM (pH 8.05) or PBS (pH 7.9) was added on top of each gel. 1% v/vantibiotic solution (penicillin and streptomycin) was added to the DMEMto prevent bacterial contamination. All samples were maintained at 37°C. and 5% CO₂ in a humidified incubator. At the indicated time points,the solution (DMEM or PBS) was carefully removed using a 1 mLmicropipette and the gel was weighed (W_(f)). The media was carefullyreplaced on the gel and returned to the incubator at 37° C. Degradationwas monitored from the relative weight percentage of the hydrogel atdifferent time using Eqn. 3:

$\begin{matrix}{{{Relative}{weight}{percentage}{of}{polymer}} = \left( {\frac{W_{f}}{W_{0}} \times 100\%} \right)} & {{Eqn}.3}\end{matrix}$

It was observed that formulations F1, F3, and F5 (all with the lowermolar ratio of thiol-to-acrylate of 1.0) degraded faster than F2, F4,and F6 (molar ratio of 1.05) in both DM EM (FIG. 4A) and PBS (FIG. 4B).Interestingly, all formulations exhibited greater stability (defined bymaintaining the relative weight percent) in PBS with F6 containing ˜100%of its initial weight for up to 400 h in PBS compared to 60 h in DMEM (a6-fold increase in stability). Additionally, the decrease in relativeweight was much steeper in hydrogels incubated with PBS compared tothose incubated with DMEM. This suggests a slow degradation under cellculture conditions and a fast degradation in salt buffered solutions.

Similar to the findings for gelation time, the monomer weight percentageand molar ratio of thiol to acrylate were also found to modulate theswelling, degradation, and stiffness of the hydrogel. Increasing themonomer content from 8.5 to 9.5 wt % and the molar ratio of thiol toacrylate from 1.0 to 1.05 resulted in higher swelling ratios and longerdegradation time (FIGS. 2B and 4A-4B). Due to an increase in the polymercontent in the hydrogel, there was an increase in the crosslinkingdensity, which decreased the gelation time and increased the degradationtime. This is supported by the fact that the storage modulus (G′) islinearly dependent on the crosslinking density of the polymer. The datapresented here (FIGS. 5 and 11A-11B) showed a linear increase in G′ andG* from F1 to F6, which suggest that crosslinking density increases withincreasing polymer weight percentage. This can explain why F6 takeslonger to degrade than the other formulations. Changes in hydrogelswelling ratio and degradation time due to increasing the molar ratio ofthiol to acrylate from 1.0 to 1.05 can be explained by threepossibilities: (a) an increasein polymer crosslink probability, (b)uncrosslinked thiol groups remaining in the hydrogel (FIG. 1 b , ii, and(c) potentially impure thiol compounds. A comparison between F1 and F2(same weight percentage, different molar ratios) shows a greaterswelling and decreased degradation in F2. Moreover, F2 exhibits agreater degree of crosslinking than F1 based on a higher value for G′(FIGS. 5 and 11A-11B). These findings indicate that adding a smallamount of excess thiol (e.g., increase the molar ratio) maximizes thereaction conversion coupled with some remaining free thiol groups thatincrease hydrogel mesh size to enhance swelling. The rapid degradationand very low cellular viability in the molar ratio of hydrogels of 1.0(F1, F3, and F5) indicate that it may be possible that ETTMP is not 100%pure, resulting in a not fully crosslinked hydrogel. This results in apotential remainder of excess acrylate groups that over time decomposesto acrylic acid, which can explain poor cell viability in both the 2Dand 3D culture studies (FIGS. 6, 7, and 12 ) Additionally, incubation ofhydrogels in a buffer with a higher pH (DMEM) resulted in a fasterdegradation time, which can be attributed to increased ester hydrolysisat higher pH values. Therefore, the weight percentage of polymer, molarratio of thiol-to-acrylate groups, and pH of the media provides controlover degradation of the thiol-acrylate hydrogel.

Rheology

Precise control of polymer stiffness is essential for any biological orsynthetic hydrogel used for 3D cell culture applications. Numerousstudies have shown that the stiffness of the hydrogel can alter cellproliferation and viability. As such, it was important to measure therelative stiffness for all six formulations of the thiol-acrylatehydrogels. Rheology measurements were performed by implementing a smallamplitude oscillatory shear using an 8 mm parallel disk geometry at 37°C. for a frequency range of 0.682 to 62.8 radians per second and aconstant shear strain amplitude of 10%. The complex shear modulus (G*)value was calculated using Eqn. 4:

|G*|=√{square root over ((G′)²+(G″)²)}  Eqn. 4

G′ is the true or shear storage modulus which describes the polymerresistance to deformation and G″ is the imaginary modulus, or lossmodulus, which provides information about the loss of polymer mechanicalenergy though dissipation of heat. The stiffnesses of the differenthydrogel formulations were compared from the values of G* and thetangent of the phase angle (δ) difference between the applied stress andthe deformation (strain). The complex shear modulus |G*| providedinsight into the gel stiffness under dynamic conditions. Tan δ wascalculated using Eqn. 5:

$\begin{matrix}{{\tan\delta} = \frac{G^{''}}{G^{\prime}}} & {{Eqn}.5}\end{matrix}$

If tan δ (0≤tanδ1) is around zero, the material is purely elastic but iftan δ is around one or greater than one, then the material is a viscousliquid,

Using bulk rheology, it was found that F6 (with the highest weightpercentage of monomers) exhibited the greatest degree of stiffness whileF1 was found to be the least stiff hydrogel (FIG. 11A-11B). This wasdetermined by the separation between shear storage modulus (G′) and lossmodulus (G″) resulting in the value of the complex shear modulus (G*)being the highest (and tan δ being the lowest) for F6, which is oppositefor F1 (FIG. 6 ). Similarly, the observed stiffness was found todecrease from F6 to F1 (with decreasing weight percentage of monomer).Increasing the molar ratio of thiols to acrylate groups (1.0 to 1.05)was also found to increase the stiffness of the corresponding hydrogelsignificantly. This indicates that hydrogel stiffness depends upon boththe weight percentage of monomer in the hydrogel as well as the molarratio of thiol to acrylate, with a greater amount of thiol providing astiffer hydrogel (comparing F2 to F1).

Example 4 Visualization of Exemplary Hydrogels

2D visualization of Cell Adhesion, Proliferation, and Viability

Once all of the chemical and physical properties of the thiol-acrylatehydrogels were determined, the next step was to evaluate how theyfunctioned as a scaffold to support cancer cell proliferation. Initialassessment of the cytotoxicity of the thiol-acrylate hydrogels wasperformed using a 2D cell culture approach to eliminate any questionsregarding mass transfer of nutrients into the hydrogel. 100 μL of eachhydrogel formulation was added to wells of a 96 well plate (Corning).After the hydrogel solidified, 100 μL of a suspension of MDA-MB-231cells (2.5×10⁴ cells/mL) in complete media was added on top of thehydrogel. Cells were cultured at 37° C. with 5% CO₂ in a humidifiedincubator for nine days. To ensure sufficient nutrients, complete mediawas replenished every two days. Cell viability was determined after ninedays of culture using the live cell stain Calcein AM (Life Technologies)and the dead cell stain ethidium homodimer-1 (EthD-1, Life Technologies)at concentrations of 3.5 μM and 3.85 μM, respectively, in extracellularbuffer (ECB). At day nine, the media was removed from each well followedby immediate addition of 100 μL of staining solution, which wasincubated with the cells for 70 min in the dark followed byvisualization using fluorescent microscopy. Cellular fluorescence wasvisualized using a Leica DMi8 inverted microscope outfitted with a FITCfilter cube, 20× objective (Leica HC PL FL L, 0.4× correction), andphase contrast and brightfield applications. Digital images wereacquired using the Flash 4.0 high speed camera (Hamamatsu) with a fixedexposure time of 35 ms for the FITC filter (green, live cells), 55 msfor the rhodamine filter (red, dead cells), and 10 ms for brightfield.Image acquisition was controlled using the Leica Application Suitesoftware. All images were recorded by using the same parameters. Acontrol experiment was performed using 3 wt % agarose (in DI water) gelinstead of the thiol-acrylate hydrogels. For this experiment, 100 μL ofwarm agarose was added to the 96 well plate followed by the same methoddescribed above.

Initial assessment of the cytotoxicity of the thiol-acrylate hydrogelswas performed using a 2D cell culture approach to eliminate anyquestions regarding mass transfer of nutrients into the hydrogel.Triple-negative breast cancer cells (MDA-MB-231), model cancer cellline, were seeded on top of all six hydrogel formulations and allowed toadhere and grow for nine days at 37° C. followed by a terminal viabilitystain (FIG. 6 ). A relationship between cell attachment (andproliferation) and molar ratio of thiol to acrylate groups was observed.All three formulations with the 1.05 molar ratio (F2, F4, and F6)supported the attachment and proliferation of the cancer cells (FIG. 6). Analysis of the cells found that 95-97% of cells were viable on F2,F4, and F6. Conversely, the cancer cells were unable to adhere to thesurface for all three formulations with the 1.0 molar ratio (F1, F3, andF5) resulting in an aggregate suspension of cells above the surface ofthe hydrogel. The majority of the cells incubated with F1, F3, and F5were also found to not be viable after nine days of culture with ˜1-5%viable cells remaining (FIG. 6 ). This suggests that the 1.0 molar ratioof thiol to acrylate not only prevented attachment, but also wascytotoxic. To ensure it was the thiol-acrylate hydrogel that resulted incell death rather than the method, a control 2D proliferation experimentwas performed using agarose as the basement hydrogel. As expected, thecells were not able to adhere to the agarose, but were found to beviable after nine days of culture, further confirming that theformulations with the 1.0 molar ratio of thiol to acrylate (F1, F3, andF5) were cytotoxic.

This suggests that the thiol-acrylate hydrogels with the 1.05 molarratio of thiols to acrylates is compatible with cell spreading and thatcancer cells can release their own extracellular matrix (ECM) componentson the hydrogels to facilitate proliferation. Finally, there was noobservable difference in cell viability or proliferation in thedifferent weight percentage formulations.

Results from the studies presented herein confirm that the threeformulations using the molar ratio of thiol to acrylate of 1.05 werenoncytotoxic and capable of supporting cellular adhesion and growthwhile maintaining sufficient viability both two dimensionally and threedimensionally. This is supported by the observation of very little celldeath of MDA-MB-231 cells grown in 3D (FIGS. 6 and 13 ) and theformation and growth of a significant number of MCF7 3D spheroids (FIGS.8 and 14 ) in the formulations with the excess thiol (F2, F4, and F6).All these findings indicate that for 3D cell culture in these types ofgels, two factors affect cellular growth: the molar ratio of thiol toacrylate and weight percentage of polymer in hydrogel formulation.

3D Visualization of Cell Proliferation and Viability

Proliferation and viability of cancer cells in the thiol-acrylatehydrogels grown under 3D conditions were assessed. MDA-MB-231 cells weredetached from the T-75 flasks and centrifuged at 1800 rpm for 2.5 min asdescribed previously. The supernatant was removed and the cell pelletwas resuspended in the pre-mixed thiol-acrylate hydrogel to a finalvolume of 1 mL with a final cell density of 5×10⁴ cells/mL. All sixhydrogel formulations were prepared as described above with the vortexstep occurring prior to the addition of the cells to avoid cellularshearing. The cell/hydrogel suspension was mixed gently using amicropipette to ensure a homogeneous distribution of cells within thehydrogel. An advantage of the thiol-acrylate hydrogel is that thetime-dependent gelation by the base catalyzed Michael addition allowsfor manipulation and transfer of the suspension for ˜2-8 min tofacilitate the transfer of the suspension to cell culture plates. 100 μLof the cell/hydrogel suspension was transferred to each well of a96-well plate specifically designed for the growth of 3D spheroids(Corning 4515). Once the gel solidified, 100 μL of cell culture mediawas added to each well and the cells were incubated as describedpreviously. The growth media was replenished every two days to ensureproper nutrient levels. Assessment of cellular viability was performedas described above on days 4, 7, 11, and 17. The number of live and deadcells were calculated using a custom Python code called FluoroCellTrack.MCF-7 was seeded into TA hydrogels with excess thiol (F2, F4, and F6) asdescribed earlier for MDA-MB-231 cells with a cell density of 1×10⁵cells/ml. Brightfield images were acquired every 24 hr to evaluatespheroid formation and their growth.

Here, MDA-MB-231 cells were immediately seeded into all six formulationsof the thiol-acrylate hydrogel and allowed to grow for 17 days in acustom spheroid 96-well plate at 37° C. The findings from thisexperiment were similar to what was discovered with the 2D proliferationstudies; however, the weight percentage of the monomers in the hydrogelinfluenced growth and proliferation Cells seeded within the formulationswith the 1.05 molar ratio of thiol to acrylate and the lower weightpercentages (F2: 8.5% and F4: 9%) were observed to grow and remainviable after 17 days of culture (FIG. 7 ). Moreover, in F2 and F4 thecells formed into the aggregates with a mix of alive and dead cells.Interestingly, the highest weight percentage (9.5%) formulation usingthe 1.05 molar ratio (F6) exhibited the lowest overall viability after17 days (FIG. 7 ) suggesting that higher monomer content, correspondingto the stiffest hydrogel, did not provide optimal 3D cellular growthconditions. Similarly to what was observed in the 2D proliferationstudies, none of the 1.0 molar ratio formulations (F1, F3, and F5) wereable to sustain cellular growth under 3D cell culture conditions (FIGS.7 and 12 ). To quantify the number of viable cells in all sixformulations, the microscopy data was processed by an automated Pythonalgorithm capable of identifying and counting fluorescent cells usingdifferent channels (e.g., green for live and red for dead). The numberof viable cells observed in all six formulations throughout the 17-daytime course is consistent with the microscopy data presented in FIG. 7 .To quantify the number of viable cells in all six formulations, themicroscopy data were processed by an automated Python algorithm capableof identifying and counting fluorescent cells using different channels(e.g., green for live and red for dead). FIG. 13 summarizes the numberof viable cells observed in all six formulations throughout the 17-daytime course, which is consistent with the microscopy data presented inFIGS. 7 and 12 . Additionally, it was observed that cancer cells thatwere seeded closer to each other on day 0 were more likely to survive inF2 and F4 due to their ability to communication with nearby cells andeventually form aggregates. MCF7 spheroids growing on a hydrogeldisclosed herein can be seen in FIG. 15 . Conversely, single isolatedcancer cells were more likely to be non-proliferative and dead by theend of the 17-day period.

The results from the MDA-MB-231 studies above confirmed the growth andviability of cancer cells in the TA hydrogels; however, this cell linehas been shown in the literature to be resistant to form 3D spheroidsand is normally referred to as cellular aggregates instead of cellularspheroids. To study this phenomenon, MCF7 breast cancer cells were usedto investigate the potential for the TA hydrogel to support 3D spheroidformation. This cell line has been well documented to form 3D spheroidsusing a number of methods and materials. MCF7 cells were seeded in thethree formulations with a higher thiol-to-acrylate ratio (F2, F4, andF6), which demonstrated the best viability with the MDA-MB-231 cells.The cells were incubated for 12 days at 37° C. to observe 3D spheroidformation. It was observed that all three formulations supported theformation and growth of 3D spheroid of -20-25 spheroids per well (FIG. 8). 3D spheroids started to form by Day 2 in F2 and by Day 3 in F4 andF6. The diameter of the spheroids increased with time during the 12-dayincubation, indicating significant cellular growth. After 12 days ofculturing, 3D spheroids with diameters ranging from 100 to 600 μm wereobserved. Additionally, the 3D spheroids were observed to aggregate inthe center of the well during the 12-day incubation due to thetime-dependent degradation of the TA hydrogel.

Example 5 Microfluidic Device Preparation and Viability Studies

Microfluidic devices have become a tool to rapidly generate 3Dspheroids. A popular approach utilizes microwell arrays that have beenmodified to prevent cellular attachment and force cellular aggregationinto 3D spheroids. These devices can rapidly generate a large number ofspheroids; however, most microwell arrays cannot facilitate on-chipinterrogation of 3D spheroids and suffer from disaggregation due tofluid shear stress. Similarly, the spheroids generated in the abovemethods suffer from significant heterogeneity since they are generatedfrom hundreds to thousands of different cells. Described herein aremethods and systems incorporate the thiol-acrylate (TA) hydrogelscaffolds described above into a microfluidic droplet trapping array togenerate and study 3D spheroids.

Three different trapping arrays were fabricated with 70, 150, or 300 μmcircular traps to study the effect of droplet size and cell seedingdensity on spheroid formation and growth in the TA hydrogel scaffold.The 70, 150, and 300 μm trapping array consisted of 785, 990, and 450traps respectively, and was capable of ˜99% droplet trapping and ˜90%cellular encapsulation. The TA hydrogel allowed for rapid (˜30-40 min)polymerization of the scaffold followed by removal of the oil phase andreplacement with complete media to initiate spheroid growth. The growthand viability of model breast cancer spheroids using MCF7 at 37° C. wasconfirmed for up to four days under static conditions and longer withcontinuous infusion of fresh growth media. This study also identifiedthat a minimum number of encapsulated cells (˜4-6) are needed togenerate a spheroid and that single encapsulated cells are less likelyto grow into a full-blown spheroid. The findings from this studyhighlight an alternative approach to generate 3D spheroids incorporatingan easy-to-use, inexpensive scaffold that can be used forhigh-throughput drug screening.

Device Preparation

The device described herein includes a flat layer having two inlets andan outlet. The flat layer is above a bottom PDMS device layer thatincludes microfluidic flow channels and a trapping array, such the thatthe trapping array is provided below the flow channels (FIG. 15 ).

The PDMS layer, or device replica, can be formed from a silicon master.Silicon wafers for different trapping sizes were developed usingstandard soft lithography. In some aspects, the wafers are about 4 inchwafers. Briefly, AutoCAD (2015 version, AutoDesk) was used to creategeometries for the microchannels which were printed onto ironoxide/chrome masks (Front Range). A silicon master was fabricated usinga two-step lithography process to generate the bottom fluidic layer andtop trapping array. SU-8 2025 (MicroChem) was spun onto a clean 4″silicon wafer (University Wafer) using a spin coater (WS-650 Series SpinProcessor, Laurel) Technologies Corp) at 3000 rpm for 30 s to achieve athickness of 40 μm for the bottom fluidic layer. The wafer was pre-bakedat 65° C. for 5 min and then baked at 95° C. for 25 min followed by agradual cooldown to 25° C. UV exposure was performed in a custom-builtUV exposure system with a Blak-Ray B-100 series UV lamp (UVP, LLC) with1 mW/cm² power intensity for 60 s. A post exposure bake was performed at65° C. for 5 min and at 95° C. for 25 min. A second 40 μm layer of SU-82025 was spun onto the wafer to generate the overhead trapping arrayfollowed by the same pre-exposure bake, UV exposure, and post-exposurebake steps. Following the second post exposure bake step, the wafer wasimmersed in an SU-8 developer solution (MicroChem) for -5 min followedby a rinse with isopropyl alcohol (VWR) to remove all uncrosslinkedSU-8. The wafer was dried with compressed nitrogen and then hard bakedat 150° C. for 30 min to stabilize the patterns.

To make the Polyclimethylsiloxane (PDMS) device replica, a base andcuring agent were mixed in a ratio of 10:1 and degassed under vacuum for45 minutes before pouring it on a silicon wafer. Similarly, to obtain aflat PDMS layer 20 g of degassed PDMS mixture (having the same ratio ofbase and curing agent as before) poured on a 100 mm petri dish andplaced it on a hot plate at 65° C. for 12 hours to cure completely. ThePDMS replica and flat layer were carefully removed from the wafer andpetri dish before cutting them in proper sizes using the X-Acto knife.The PDMS flat layer and device replica were aligned visually to makeholes from the flat layer side at two inlets and outlet using a blunted18-gauge needle. Later, those holes were blown with high-pressurenitrogen to remove any unwanted tiny PDMS pieces that were stuck insidethe inlets and outlet holes. Both layers of PDMS were thoroughly cleanedusing scotch tape before binding them using a plasma binder. The plasmabinder was vacuumed for 2 minutes and 30 seconds before treating thePDMS layers with plasma for 55 seconds. Finally, both the PDMS devicereplica and flat layer were aligned visually and left in a petri dish atroom temperature overnight to develop strong interaction between thesetwo layers. The surface inside the device was made hydrophobic bytreating the inside of the device with aquapel and excess aquapel wasremoved by flowing high-pressure nitrogen gas and Novec 7500 oil throughthe device.

Working Principle of Droplet Generator

The device was wiped with a chem wipe socked with 70% (v/v) ethanolsolution and once dried, it was placed on a sterile 100×20 mm petridish. Six 16-inch-long microfluidic tubes were cut and autoclaved beforeusing them in a different stage of the on-chip experiment. The dropletgenerator used in this system has two inlets: one is used to inject oiland surfactant mixture and the other inlet is used to inject hydrogelmixed with cells (FIG. 2A) into the device. Before starting, the dropletgeneration air inside the device was removed by flowing Novec 7500 oilcontaining 0.5% (w/w) Fluro-surfactant at a flow rate of 1000 μL/h.While air from the device was removed, MCF-7 cells were centrifuged at300 g for 6 minutes to obtain a cell pellet. In the meantime, 8.5 wt. %thiol acrylate (TA) hydrogel was made in Dulbecco's Modified EagleMedium (DMEM) media (containing 1% v/v penicillin-streptomycin) aspreviously described. Both hydrogel and the test tube containing cellswere brought under a biosafety hood. The cell pellet was mixed with 1 mLof liquid hydrogel mixture using a 1 mL micropipette and transferredinto a 1 mL sterile syringe connected with a 23-gauge needle underneaththe biosafety hood. To make hydrogel droplets, both oil containing 0.5%(w/w) fluorosurfactant and hydrogel were flowed at a specific flow rate(Table 3) using two Harvard syringe pumps which generate droplets at theflow-focusing junction of the device (FIG. 16A).

TABLE 3 Droplet generator parameters tested for spheroid growth To getappropriate Cell droplet size Fluidic seeding Experi- Oil and Gel TrapTrap layer density mental surfactant flow diameter height height(million length flow rate rate (μm) (μm) (μm) cells/mL) (Days) (μL/h)(μL/h) 70 40 40 2.5 3 40 70 150 150 100 3 to 4 7 550 750 300 3 to 4 7200 750 300 100 40 3 3 200 70 300 100 3 7 150 700

Due to the oil flow, generated droplets are carried out to the trappingarray and trapped into the traps (FIG. 16B). Once all the traps werefilled with droplets, droplet generation was turned off and any extradroplet from the trapping was flushed away from the device using oilflow (1000 uL/h). Followed by the removal of extra droplets, the syringewas swapped with a syringe containing only Novec 7500 oil. The oil wasflowed at a rate of 500 uL/h for one hour before introducing media intothe device to remove surfactant from the device, as the presence ofsurfactant negatively effects later steps such as removal of oil fromthe device using media flow. During this one-hour waiting period, theMichael addition reaction between thiolate and acrylate was performedand resulted in fully crosslinking hydrogel. During the completion ofgelation, the whole device was submerged beneath autoclaved deionized(D1) water containing 1% (v/v) penicillin-streptomycin to preventdroplets from shrinking due to water evaporation and to prevent air fromentering the device. The media was introduced and flowed through thedevice at a rate of 500 uL/h for 30 minutes, then the whole device wasimaged at 5× magnification under a Leica DMi8 inverted microscope.

Finally, the water used to submerge the device was removed using a 25 mLpipet. DMEM media containing 1% (v/v) penicillin-streptomycin was addedinto the petri dish. While the device was submerged underneath the DMEMmedia, all three microfluidic tubings (inlets and outlet) were carefullyremoved, and the petri dish was covered with a lid before placing itinside the 37° C. incubator. The media was replaced every two days.However, from several trials, it was observed that beyond day twospheroids were not growing inside the device (FIGS. 17A-17C). To solvethis issue, gravity-driven media flow through the device (below) wasintroduced for continuous nutrient flow and waste removal from thedevice.

Incorporation of Gravity-Driven Media Flow to Maintain Cells Inside theDevice

Continuous nutrient flow to the cells is very important for cells tosurvive and to multiply. This continuous media flow can be providedusing syringe pumps. However, syringe pumps are expensive, hard to movefrom one place to another, and difficult to fit inside a CO₂ incubatordue to space limitations. Finally, in the syringe pump setup, it isdifficult to swap a new syringe with the old one without introducing airinside the device. However, with the gravity-driven flow, most of theseproblems can be solved. To develop this system, a single block oftest-tube holders was used as a stand to hold both the reservoir andcollector at a height of 13 and 11.5 cm, respectively. Both thereservoir and collector were made of 5.0 mL BD plastic syringes and a23-gauge needle was used to connect the microfluidic tubing with thesyringes. Microfluidic tubing from the reservoir was connected to theoil port of the device, which helped media to flow through the devicedue to pressure difference between the reservoir and collector. Finally,the media exited through the outlet port, which was connected to thecollector by another piece of microfluidic tubing. To prevent any medialeakage through the aqueous port, it was closed by connecting a closedsyringe using microfluidic tubing. For cell culture purposes, media wasreplenished every 24 hours by adding 4 mL media into the reservoir andat the same time removing 3 mL from the collector (FIGS. 18 and 19 ).

The amount of media in the reservoir and space between the reservoir andcollector effects the resultant flow rate and can be adjusted on thedevice according to need. The ratio of fluidic layer depth and trapheight can be tuned to prevent cells escaping from their traps. In oneexample, the microfluidic trapping array can have a 150 μm tap diameter,300 μm trap height, and 100 μm tall microfluidic layer.

On-Chip Viability Study

Live and dead staining were carried out after nine days of on-chip cellculture using the live stain Calcium AM (Life Technologies) and the deadstain Ethidium homodimer-1 (EthD-1, Life Technologies) of concentrations3.75 μM and 4.5 μM respectively in ECB. Live and dead stain mixtureswere flowed through the device at a rate of 350 μL/h for 1 hour and 45minutes using a Harvard syringe pump. The device was incubating at 37°C. during the whole time of stain mixture flow. Finally, cellularfluorescence was visualized using a Leica DMi8 inverted microscopeoutfitted with a FITC filter cube, rhodamine filter, and brightfieldapplications at 5× objective. Digital images were acquired using theFlash 4.0 high-speed camera (Hamamatsu) with a fixed exposure time of 15ms for the FITC filter (green, live cells), 100 ms for the rhodaminefilter (red, dead cells), and 20 ms for brightfield. This on-chipviability study shows that most of the cells are living with few dead(FIGS. 20A-20E). This indicates that the device is cell-culturecompatible, and with correct protocols, it can be used for rapid andhigh throughput spheroid generation.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations setforth for a clear understanding of the principles of the disclosure.Many variations and modifications may be made to the above-describedembodiment(s) without departing substantially from the spirit andprinciples of the disclosure. All such modifications and variations areintended to be included herein within the scope of this disclosure andprotected by the following claims.

What is claimed is:
 1. A system for 3D cell culture comprising: amicrofluidic droplet-generating device and a tunable cell culturematerial comprising a thiol-acrylate hydrogel.
 2. The system of claim 1,wherein the microfluidic droplet-generating device comprises a droplettrapping array located below a microfluidic flow channel, wherein thetrapping array comprises a plurality of circular traps.
 3. The system ofclaim 2, wherein the circular traps have a diameter of about 70 μm toabout 300 μm.
 4. The system of claim 1, wherein the thiol-acrylatehydrogel is a product of a reaction between a thiol comprisingethoxylated trimethylolpropane tri (3-mercapto-propionate) (ETTMP), andan acrylate comprising poly(ethylene glycol) diacrylate (PEGDA); andwherein the reaction is a base-catalyzed Michael addition occurring at apH of about 7.6 to 8.2.
 5. The system of claim 4, wherein thethiol-acrylate hydrogel and cells are provided to the microfluidicdroplet-generating device in an aqueous phase, and the reactioncompletes polymerization inside a trapping array.
 6. The system of claim1, wherein: when a hydrogel weight percent is about 8.5 and a molarratio is about 1.0, a gelation time is about 150 minutes; when thehydrogel weight percent is about 8.5 and the molar ratio is about 1.05,the gelation time is about 40 minutes; when the hydrogel weight percentis about 9 and the molar ratio is about 1.0, the gelation time is about80 minutes; when the hydrogel weight percent is about 9 and the molarratio is about 1.05, the gelation time is about 30 minutes; when thehydrogel weight percent is about 9.5 and the molar ratio is about 1.0,the gelation time is about 30 minutes; and when the hydrogel weightpercent is about 9.5 and the molar ratio is about 1.05, the gelationtime is about 25 minutes.
 7. The system of claim 3, wherein the trappingarray has about 785 circular traps having a diameter of 70 μm each. 8.The system of claim 3, wherein the trapping array has about 990 circulartraps having a diameter of 150 μm each
 9. The system of claim 3, whereinthe trapping array has about 450 circular traps having a diameter of 300μm each.
 10. The system of claim 1, wherein the microfluidicdroplet-generating device comprises a flat layer having two inlets andan outlet, wherein the flat layer is above a bottom PDMS device layerthat includes the microfluidic flow channel and the droplet trappingarray, such the that the droplet trapping array is provided below themicrofluidic flow channel.